In Vitro Probiotic Characterization and Whole-Genome-Based Safety Screening of Bacillus spp. Isolated from the Gastrointestinal Tract of Betta splendens Regan

Spore-forming Bacillus probiotics are widely used in aquaculture, but host-associated strains from Siamese fighting fish (Betta splendens Regan) remain uncharacterized. Ten Bacillus isolates were recovered from the gastrointestinal tract of farmed B. splendens by heat selection and were identified by biochemical testing and 16S rRNA gene sequencing. In vitro assays covered growth under acid, bile salts and gastrointestinal enzymes, hydrophobicity, auto-aggregation, β-galactosidase activity, antibiotic inhibition zones and antimicrobial activity. BM5 showed the highest relative growth at pH 3 and with gastrointestinal enzymes, while BM2 had the highest in 0.3% bile salts and β-galactosidase activity (p < 0.05). BF2 and BM2 were strongly hydrophobic. Measurable inhibition zones were recorded for all isolate–antibiotic combinations except BF3 with vancomycin. Notably, only BF1 inhibited Pseudomonas aeruginosa. BF1 and BM2 were selected for whole-genome sequencing. Both genomes harbored intrinsic chromosomal resistance genes characteristic of the B. pumilus group (blaBPU-1-like and cat86), neither of which was flanked by an insertion sequence. Homologues of 35 virulence-associated genes, including hlyIII and inhA, were identified. Neither acquired resistance determinants nor genes encoding enterotoxins or emetic toxins were detected in the database searches. However, these database-dependent findings are only a preliminary screen and do not establish safety; in vivo evaluation is warranted.